Standardized mitochondrial analysis gives new insights into mitochondrial dynamics and OPA1 function
Arnaud ChevrollierJulien CassereauMarc FerréJennifer AlbanValérie Desquiret‐DumasNaïg GuéguenPatrizia Amati‐BonneauVincent ProcaccioDominique BonneauPascal Reynier
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MFN2
Mitochondrial disease
DNAJA3
Mitochondria change their shape dynamically, mainly
through fission and fusion. Dynamin-related GTPases have been shown
to mediate remodeling of mitochondrial membranes during these
processes. Mitochondrial fission in mammals is mediated by the
dynamin-like protein DLP1/Drp1 that is recruited to the outer
mitochondrial surface through the membrane-anchored protein hFis1.
Another GTPase, Mitofusin (Mfn), is anchored at the outer
mitochondrial membrane and mediates fusion of the outer membrane.
Mammalian cells have two Mfn isoforms, Mfn1 and Mfn2, that share a
conserved molecular structure. Either Mfn1 or Mfn2 can functionally
replace each other in Mfn-null cells, suggesting their conserved
role as fusion proteins as well. This thesis research centers on
the mitochondrial fusion protein Mfn2. Specifically, it focuses on
studying the effect of the Mfn2-induced mitochondrial shape change
on mitochondrial function and the molecular mechanisms of
mitochondrial fusion mediated by the Mfn2 protein.
We found that
overexpression of Mfn2 drastically changes mitochondrial
morphology, forming mitochondrial clusters. High-resolution
microscopic examination indicated that the mitochondrial cluster
consisted of small fragmented mitochondria. Inhibiting
mitochondrial fission prevented the cluster formation, supporting
the notion that mitochondrial clusters are formed by
fission-mediated mitochondrial fragmentation and subsequent
aggregation. Mitochondrial clusters displayed a decrease in inner
membrane potential and proton pumping activity, suggesting
functional compromise of small fragmented mitochondria by Mfn2
overexpression; however, mitochondrial clusters still retained
mitochondrial DNA. We found that cells containing clustered
mitochondria lost cytochrome c from mitochondria and underwent
caspase-mediated apoptosis. These results demonstrate that
mitochondrial deformation impairs mitochondrial function, leading
to apoptotic cell death and suggest the presence of an intricate
form-function relationship of mitochondria. Because intra- and
inter-molecular interactions play an important role in the membrane
remodeling action of dynamin family proteins, we analyzed domain
interactions of the Mfn2 molecule using genetic and biochemical
approaches. We found that two hydrophobic heptad-repeat (HR)
domains, HR1 and HR2, interact with each other, in addition to the
already reported HR2 and HR2 interaction. Interestingly, we
discovered that the region of Mfn2-HR1 interacting with HR2 also
interacts with the C-terminal coiled-coil domain of the fission
protein DLP1 (DLP1-CC). We identified mutations in the Mfn2-HR1
region that selectively disrupt the HR1/HR2 interaction and the
Mfn2/DLP1 interaction. Morphological analyses indicated that the
Mfn2/DLP1 interaction participates in mitochondrial fusion whereas
the association of HR1 and HR2 of Mfn2 is inhibitory in the fusion
process. These data suggest that DLP1 functions as a regulatory
factor interacting differentially with Mfn2 and hFis1, which
provides a novel mechanism for efficient…
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Abstract Mitochondrial structural dynamics is regulated by the fusion or fission of these organelles. Recently published evidence indicates the vital role of mitochondrial fusion and fission in cellular physiology, including progression of apoptosis. These reports indicate that in addition to intimate link between mitochondrial morphogenesis machineries and regulation of mitochondrial steps in apoptosis, certain proteins vital for the regulation of mitochondrial steps in apoptosis can also regulate mitochondrial fusion and fission in healthy cells. In this article, we focus on the regulation of mitochondrial network dynamics. The emerging evidence indicating that proteins implicated in mitochondrial network dynamics are vital for the mitochondrial steps in apoptosis is presented here, as well. Furthermore, the data demonstrating an unexpected role for the B‐cell lymphoma (Bcl)‐2 family members in the regulation of mitochondrial morphogenesis are also discussed. Key concepts: In healthy cells, mitochondrial cycle between several shapes, their morphology result from the equilibrium between mitochondrial fusion and fission. The unique feature of mitochondrial fusion is the necessity of merging double membrane systems from the two fusing mitochondria. This process is mediated by the outer mitochondrial membrane‐associated mitofusin proteins (Mfn1 and Mfn2), and the inner mitochondrial membrane‐associated Opa1. Regulation of mitochondrial fusion and fission has a significant impact on cell viability and early development. The mitochondrial fragmentation occurs concomitantly with the outer mitochondrial membrane (OMM) permeabilization, a critical step in apoptosis. The cooperation between proteins involved in mitochondrial fusion and fission and Bcl‐2 family proteins during apoptosis suggests that changes in mitochondrial network dynamics contribute to apoptotic signalling. The mechanistic link between the core mitochondrial fusion and fission regulating proteins (e.g. Drp1, Mfn2 and Opa1) and proteins from Bcl‐2 family, suggest that Bcl‐2 family proteins also regulate mitochondrial dynamics in healthy cells.
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Mitochondria play a key role in maintaining cellular metabolic homeostasis. These organelles have a high plasticity and are involved in dynamic processes such as mitochondrial fusion and fission, mitophagy and mitochondrial biogenesis. Type 2 diabetes is characterised by mitochondrial dysfunction, high production of reactive oxygen species (ROS) and low levels of ATP. Mitochondrial fusion is modulated by different proteins, including mitofusin-1 (MFN1), mitofusin-2 (MFN2) and optic atrophy (OPA-1), while fission is controlled by mitochondrial fission 1 (FIS1), dynamin-related protein 1 (DRP1) and mitochondrial fission factor (MFF). PARKIN and (PTEN)-induced putative kinase 1 (PINK1) participate in the process of mitophagy, for which mitochondrial fission is necessary. In this review, we discuss the molecular pathways of mitochondrial dynamics, their impairment under type 2 diabetes, and pharmaceutical approaches for targeting mitochondrial dynamics, such as mitochondrial division inhibitor-1 (mdivi-1), dynasore, P110 and 15-oxospiramilactone. Furthermore, we discuss the pathophysiological implications of impaired mitochondrial dynamics, especially in type 2 diabetes.
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Mitochondria are frequently described as the powerhouse of the cell for apparent reasons. However, these organelles are dynamic was not known until recently. Scientists have found that mitochondria must undergo well-organized cycles of fragmentation/fission and fusion to maintain structural integrity, size, and distribution. These fission and fusion events are collectively called “mitochondrial dynamics” and are considered crucial for regulating organelle function. Mitochondrial fission accounts for the division of one mitochondrion into two. It is regulated by GTPase dynamin-related protein 1 (DRP1) and its adaptor proteins such as mitochondrial fission protein 1 (FIS1), mitochondrial fission factor (MFF), and mitochondrial dynamics protein of 49 and 51 kDa (Mid49, Mid51). DRP1, a cytosolic protein, is recruited to mitochondria to cause fragmentation upon activation through upregulation of serine 616 and downregulation of serine 637 phosphorylation. In contrast, mitochondrial fusion involves the fusion of two separate small mitochondria into one large mitochondrion, thereby generating a network of elongated or tubular mitochondria. These fusion events are regulated by GTPase dynamin-like proteins located on the outer (Mitofusin 1, MFN1 and mitofusin 2, MFN2) and inner (optic atrophy protein 1, OPA1) mitochondrial membrane. Fission is generally coupled with apoptosis, while fusion is associated with pro-survival signals. However, cancer cells can utilize mitochondrial dynamics, depending on their cellular state; this is reflected in the current conflicting literature explaining mitochondrial fission or fusion influencing tumor progression. Nonetheless, alterations in mitochondrial dynamics have been implicated as one of the key factors in tumor progression and therapeutic resistance across a wide spectrum of cancers. As a result, targeting mitochondrial dynamics is emerging as a potential strategy for solid tumors.
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In animals, mitochondria are mainly organised into an interconnected tubular network extending across the cell along a cytoskeletal scaffold. Mitochondrial fission and fusion, as well as distribution along cytoskeletal tracks, are counterbalancing mechanisms acting in concert to maintain a mitochondrial network tuned to cellular function. Balanced mitochondrial dynamics permits quality control of the network including biogenesis and turnover, and distribution of mitochondrial DNA, and is linked to metabolic status. Cellular and organismal health relies on a delicate balance between fission and fusion, and large rearrangements in the mitochondrial network can be seen in response to cellular insults and disease. Indeed, dysfunction in the major components of the fission and fusion machineries including dynamin-related protein 1 (DRP1), mitofusins 1 and 2 (MFN1, MFN2) and optic atrophy protein 1 (OPA1) and ensuing imbalance of mitochondrial dynamics can lead to neurodegenerative disease. Altered mitochondrial dynamics is also seen in more common diseases. In this review, the machinery involved in mitochondrial dynamics and their dysfunction in disease will be discussed.
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Mitochondria are highly dynamic organelles that produce ATP and maintain metabolic, catabolic, and redox homeostasis. Mitochondria owe this dynamic nature to their constant fission and fusion-processes that are regulated, in part, by fusion factors (MFN1 and MFN2) and fission factors (DRP1, FIS1, MFF, MIEF1, MIEF2) located on the outer mitochondrial membrane. While mitochondrial fusion and fission are known to influence mitochondrial morphology and function, a key question is whether rebalancing mitochondrial morphology can ameliorate mitochondrial dysfunction in the context of mitochondrial pathology. In this study, we used antisense oligonucleotides (ASOs) to systematically evaluate the effects of fusion and fission factors in vitro. Free uptake by cells of fusion or fission factor ASOs caused robust decreases in target gene expression and altered a variety of mitochondrial parameters, including mitochondrial size and respiration, which were dose dependent. In Mfn1 knockout mouse embryonic fibroblasts (MEFs) and MFN2-R94Q (Charcot-Marie-Tooth Type 2 Disease-associated mutation) MEFs, two cellular models of mitochondrial dysfunction, we found that ASO-mediated silencing of only Drp1 restored mitochondrial morphology and enhanced mitochondrial respiration. Together, these data demonstrate in vitro proof-of-concept for rebalancing mitochondrial morphology to rescue function using ASOs and suggest that ASO-mediated modulation of mitochondrial dynamics may be a viable therapeutic approach to restore mitochondrial homeostasis in diseases driven by mitochondrial dysfunction.
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Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes β-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.
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Mitochondria are highly dynamic organelles in a living cell,and display continuous movement,fusion and fission to form mitochondrial reticulum.Mitochondrial fission and fusion are highly regulated by a number of proteins.Mitochondrial fission is regulated by several proteins called Dnm1p,Fis1p,Mdv1p and Caf4p in yeast and Drp1,Fis1 in mammalian.On the other hand,mitochondrial fusion is mediated by an out-membrane protein,fuzzy onions(Fzo1p)in yeast,or its homologues mitofusion 1(Mfn1)and Mfn2 in mammalian.An intermembrane protein,Mgm1p in yeast and OPA1 in mammalian,involved in inner membrane fusion.Upon apoptotic stimulation,mitochondrial reticulum become fragmented.Inhibition of this process could partially prevents the release of cytochrome c and consequently cell apoptosis.Maintaining normal mitochondrial morphology is important for mitochondrial function and mitochondrial physiology,and defects in mitochondrial fission and fusion may cause severe diseases.
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Mitochondrial apoptosis-induced channel
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